Many neurodegenerative diseases are caused by the accumulation of intracellular or extracellular proteins that form amyloid deposits. In general, amyloid deposits can cross cell membranes to spread the toxic amyloids to neighboring cells. Since this mode of propagation is similar to the seeding that occurs in infectious prion disease, prion-like transmission appears to be common to many neurodegenerative diseases. To protect against the accumulation of toxic amyloid aggregates, molecular chaperones function to disaggregate them, but when they do not maintain quality control, protein aggregates are formed. My research is at the crossroads of these two areas; examining the formation and propagation of protein aggregates and their disaggregation by molecular chaperones using yeast as a model organism. Specifically, we are examining the role of different proteins that affect prion propagation. Prions are infectious proteins that self-propagate by changing from their properly folded conformation to a misfolded amyloid conformation. The conversion to the amyloid conformation can occur either spontaneously or by seeding with the misfolded amyloid protein. Once conversion occurs, the amyloid prion protein continues to propagate from mother to daughter cells. We have been primarily studying the propagation and curing PSI+ and URE3 prions. The curing of these prions was simultaneously monitored by a plating assay and by fluorescence imaging using GFP-labeled prion proteins. In yeast with the amyloid misfolded prion protein, the GFP-labeled prion proteins form foci whereas in cured cells with the properly folded prion protein, the GFP-prion protein has diffuse fluorescence. Fluorescence imaging of the GFP-labeled prion protein enables us to follow the changes that occur to the foci during the curing process. There are many different chaperones involved in the propagation of PSI+, the aggregated self-propagating form of the Sup35. These chaperones include Hsp104, Hsp70, Hsp40 and Hsp90. The molecular chaperone Hsp104 severs the prion seeds so inhibiting its activity prevents new seed production. Paradoxically, the PSI+ prion is also cured by overexpression of Hsp104. We determined that this curing by overexpression is due to dissolution of the prion seeds, which is dependent on the trimming activity of Hsp104. Unlike severing activity of Hsp104, which reduces the size of the prion seeds while increasing their number, the trimming activity reduces the size of the prion seeds without increasing their number. To further understand this curing process, different Hsp104 constructs were overexpressed with both strong and weak PSI+ variants; the former has more seeds and less soluble Sup35 than the latter. Trimming was measured by determining the loss of GFP-labeled Sup35 foci from cells that were still PSI+. Overexpression of Saccharomyces cerevisiae Hsp104 (Sc-Hsp104) cured the weak PSI+ variants an order of magnitude faster than the strong PSI+ variants. As expected if trimming was required for overexpression, Sc-Hsp104 trimmed the weak variants much faster than the strong variants. Similarly, overexpression of the fungal Hsp104 homologs from Schizosaccharomyces pombe (Sp-Hsp104) or Candida albicans Hsp104 (Ca-Hsp104) cured and trimmed the weak PSI+ variants, but neither cured nor trimmed the strong PSI+ variants. These results show that because Sc-Hsp104 has greater trimming activity than Ca-Hsp104 and Sp-Hsp104, it cures both weak and strong PSI+ variants although it cures the latter more slowly, whereas Ca-Hsp104 and Sp-Hsp104 cure only weak PSI+ variants. We conclude that both the conformation of the PSI+ variant and the specific fungal Hsp104 homolog that is overexpressed determine the level of trimming that occurs, which in turn determines the rate and amount of curing that is caused by Hsp104 overexpression As for other chaperones that affect curing by overexpression, Ssa1, a member of the Hsp70 family, also affect has a marked effect on the trimming activity of Hsp104. Overexpression of a dominant negative Ssa1 mutant increases the trimming of the prion seeds by Hsp104, resulting in faster curing of PSI+. Therefore, Ssa1 concentration plays a critical role in determining whether Hsp104 is able to trim the prion seeds and in turn cure PSI+ yeast when it is overexpressed. In fact, overexpression of the dominant negative Ssa1 mutant is able to cure weak PSI+ variants at endogenous levels of Hsp104. Furthermore, Sti1, an Hsp70 and Hsp90 cochaperone, is also needed for trimming of the prion seeds by Hsp104. The stringent chaperone requirement to maintain PSI+ in yeast tends to make weak PSI+ variants mitotically unstable. Unlike the PSI+ prion, the URE3 yeast prion is cured by overexpression of Btn2, Cur1 or Hsp42. In the present study, using a GFP-labeled full length Ure2 protein, which retains the properties of the native protein, we examined the curing of URE3. The GFP-Ure2 has a diffuse appearance in Ure0 yeast, in which Ure2 has the properly folded conformation, whereas the GFP-Ure2 has a punctate appearance in URE3 yeast, in which Ure2 has the amyloid conformation. Interestingly, overexpression of Hsp42, Btn2, or Cur1 all produce the same change in the GFP-Ure2 fluorescence foci; the foci assemble into large cytosolic aggregates during curing, many of which are a half micron or greater. This is in spite of the fact that Cur1 is diffusely localized in the nucleoplasm, whereas both Btn2 and Hsp42coaggregrate in the cytoplasm with Ure2. It appears that Hsp42 may be acting as a scaffolding molecule to promote protein-protein interaction because the curing of URE3 by overexpression of either Btn2 or Cur1 is markedly reduced in the absence of endogenous levels of Hsp42. These results show that curing by overexpression of Hsp42, Btn2, or Cur1 causes the formation of large cytosolic Ure2 aggregates, which in turn reduces the number of seeds and impedes their passage through the narrow bud neck during cytokinesis. This leads to the curing of URE3 by asymmetric segregation with retention of the large aggregates in the mother cell. Finally, we have been examining the role of Hsp40 proteins in the curing and propagation URE3 yeast prions. Interestingly, we find that YDJ1, a member of the Hsp40 J-domain family cures URE3 by also aggregating the Ure2 seeds. Similarly, a truncated YDJ1 fragment without the substrate binding domain also cures by forming large aggregates. This aggregation process is dependent on Hsp42 and is antagonized by Sis1, another member of the Hsp40 family. Overexpression of Sis1 also inhibits the curing of URE3 by Btn2, Hsp42, and Cur1. Collectively, these data suggest that another function of Sis1 is to bind to the prion seeds, which in turn prevents their aggregation. The interplay of these chaperones shows the complexity of the cellular protein quality control networks.